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26 Comments

It depends on how good the primers for the PCR are. We used a mediocre primer, and it did have some non-specific amplification that came up later in the cycling, and was deemed negative. Before we used the Extract-N'Amp on experimental tissue, we made sure our to test different positive samples which consistently amplified early in the cycling.

Linda is correct here. If your primers are not the best primers you could use in your PCR reactions you will get non-specific products and/or primer dimer. If you ran these reactions out on a agarose gel I would expect to see a fair amount of primer dimer. You can excluded this dimer product from your SYBR Green qPCR final Ct by putting specific melt-read points in the analysis.

I have always loaded 5 ul of the RED PCR product. With the specific kit mentioned in the above video, the kit has a "clear" ²X PCR mix provided, which can still be loaded on an agarose gel, but I would add 4 ul of a loading buffer, such as Sigma's G²5²6 to the whole PCR reaction, then load 5 ul on the gel.

Usually, I recommend that if you store the extract for more than ² weeks at 4C, I take the mouse tail or tissue out. Same gŒs for long term storage at -²0C, but I would recommend taking the mouse tail out before you freeze the extract. The mouse tail will start to degrade if kept on solution for long term. This action can release more PCR inhibitors into the extract. You should be able to take the extract out of long term storage and use it in PCR reaction again with positive results.

Depending on the starting material, you may get some precipitant in the bottom of the tube after the extraction protocol in done. Do not worry about it, if you are worried about it, a quick spin down of the extract before placing an aliquot in PCR will ensure that nothing is drawn up in your sampling.

Can genomic dna be extracted a second time from tissue that has sat as a neutralized sample at 4C? We had a staffing changeover and these samples were neglected. We have purchased all new Extract-n-Amp components and would like to re-extract if possible. Thank you for your help

Melissa,
You can re-extract a sample, but it depends on how long it has sat in the neutralized extract. If the extract has been sitting at 4C for more than 6 months, I would recommend performing a new extract. The tissue dŒs continue to breakdown slightly over a long peroidoft time at 4C, thus why I always recommend taking the tissue sample out of the extract if you will be storing it at 4C longer than a month. if the tissue sample dŒs not look to bad, I would try to re-extract it, but results may vary.
Take the sample out and wash it with 70% ethanol, then perform the normal extract-n-amp protocol for whatever type of tissue it is. You can also try using the current extract as-is or dilute it in a 50/50 mix of E75²6/N3910 (this will help it inhibitors have been released into the extract).
Let me know if you need more help.

Thank you so much for the help (and the hope that these samples can be salvaged). I had decanted the neutralized extraction as soon as I got to them. There is still a good amount of undigested tissue available for re-extraction, so I will try BOTH a re-extraction and a re-amplification of the previous extract. For the re-amp from the previous extract, how much of a sample dilution would you recommend with the 50/50 mix of E75²6/N3910? Thank you once again!!

I would try a 1:10 dilution, so 5 ul of the extract and 45 ul of the 50/50 reagent mix, then 4 ul in your PCR reaction.
Just to clarify, your tissue sample dŒs not digest in the extract-n-amp method, but sitting in the reagents at 4C over a long period of time, you will see some tissue decompistion (which is natural).

Should the Neutralization Solution B be added immediately upon removal from the heat source (ie: while the sample is still quite warm) or should the sample be placed on ice or allowed to cool in some way? I had a great initial run, but my most recent run has produced bands that are quite faint in my gel, so I am just trying to figure out which step could be improved upon.

I designed the protocol so that you can do either/or with the addition of the N3910. The N3910 reagent can be added right after the 3 minute heating step or a little time afterwards. You do not have to put the extract on ice or cool it in any way. I would recommend adding the N3910 ASAP. I would not wait hours or place the extracts at 4C for a while, but there should be no real issue if you had to do that because of some unavoidable delay.
I am curious about the details of your extract though (tissue used, amount, PCR setup, primers, etc.), if I had more info on that subject I might be better able to pinpoint a possible issue. Feel free to contact me through my Company's email: scott.weber@sial.com.
Thanks

Help me:
During DNA Extration from tŒ of mouse I added Neutralization Solution B after Extraction Sol instead of tissue preparation. What should I do now? I repeat or i can recover this problem. Reply me as soon possible

You should be able to transfer tissue piece(s) to a fresh, clean tube and wash it in 70% ethanol (just vortex a bit in ethanol and then draw off the ethanol). Then you may want to cut the tissue to allow new surfaces to interact with the extraction solution/tissue prep. Now you can proceed with the extraction protocol from the beginning again (this time with Tissue Prep instead obviously). Good luck!

Melissa is correct. I would recommend taking the sample out of tube, washing in 70% EtOH, then performing the extraction procedure again. I know mouse tŒs are very small, but if you can cut just a bit off the "cut-end", it will also help the extraction process.
Also, if you did not heat the extraction at 75C yet, you could just add the tissue prep to the mix, then follow the protocol through, but don't add the N3910 when you get to that step. This method is not recommended, but if you had a sample to try it on, it would be good info.
Any other questions feel free to contact me at my Company's email: scott.weber@sial.com.
Thanks

I don't know what starting material you are using, but quantifing the extract dŒs not always give a good answer. It is a crude extract, so many things can interfere in the process. The kit was designed to take 4ul of the extract per PCR reaction (using the ²X PCR mix in the kit). The two reagents were formulated to work together, so other PCR mixes do not always give you good results. The extract will be a 50/50 mix of E75²6 & N3910, if I were to use anything as a blank I would use that mix of the two components. An average yield from a typical mousetail extract will give you ~ 4-10 ng/ul of gDNA, just for a feeler. Good Luck!